t－PA　cDNA基因在CHO细胞中的稳定表达

STABLE EXPRESSION OF t-PA cDNA GENE IN CHO CELLS

建立稳定表达人组织型纤溶酶原激活剂（ｔｉｓｓｕｅ－ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ，ｔ－ＰＡ）的细胞株。将ｔ－ｐＡｃＤＮＡ连接在真核表达载体ｐｃＤＮＡ３的ＨｉｎｄⅢ位点，构建成重组质粒ｐｃＤＮＡ３ｔＰＡ，用磷酸钙介导的贴壁细胞转染法导入哺乳动物中国仓鼠卵巢（ＣＨＯ）细胞中，Ｇ４１８筛选培养后形成阳性克隆。结果：培养液中重组ｔ－ｐＡ（Ｒｅｃｏｍｂｉｎａｎｔｔ－ｐＡ，ｒｔ－ｐＡ）活性＞６０００ＩＵ／１０６细胞ｄ－１．Ｎｏｒｔｈｅｒｎ杂交证实ｔ－ＰＡｃＤＮＡ基因的转录。高表达细胞连续传代１０次后，培液中ｒｔ－ｐＡ活性未见明显变化。结论：转染ｔ－ＰＡｃＤＮＡ基因的ＣＨＯ细胞已形成稳定的工程细胞。

PURPOSE To establish a stable cell strain secreted recombinant tissue-type plasminogen activator(rt-PA).METHODS Recombinant plasmid pcDNA3tPA was constructed by insertion of t-pA cDNA originated from pUC 18tPA into eukaryotic expression vectors pcDNA3 at Hind Ⅲsite,and transfected into Chinese hamster ovary(CHO) cells by calcium phosphate-mediated method. After screened with G418, the positive clones were selected and further purified in conditional culture medium.RESULTS t-PA activity of the medium was more than 6000 IU/106 cells d-1. The transcription t-PA cDNA was confirmed by Northern hybridization. The expression level of t-PA cDNA clone did not change until passing 10 generations of the clone.CONCLUSIONS A genetic engineering cell strain secreted rt-PA is successfully constructed.